Nucleic acid probes and methods for detecting clinically important fungal pathogens

ABSTRACT

The current invention relates to the field of detection and identification of clinically important fungi. More particularly, the present invention relates to species specific probes originating from the Internal Transcribed Spacer (ITS) region of rDNA for the detection of fungal species such as  Candida albicans, Candida parapsilosis, Candida tropicalis, Candida kefyr, Candida krusei, Candida glabrata, Candida dubliniensis, Aspergillus flavus, Aspergillus versicolor, Aspergillus nidulans, Aspergillus fumigatus, Cryptococcus neoformans  and  Pneumocystis carinii  in clinical samples, and methods using said probes.

This application is a divisional of application Ser. No. 09/662,462,filed Sep. 15, 2000 (allowed) now U.S. Pat. No. 6,858,387, which is acontinuation of PCT/EP00/04714, filed 24 May 2000, which designated theU.S., and claims the benefit of U.S. Provisional Application No.60/138,621, filed 11 Jun. 1999, and EP 99870109.8, filed 28 May 1999,the entire contents of each of which being incorporated herein byreference.

FIELD OF THE INVENTION

The current invention relates to the field of detection andidentification of clinically important fungi. More particularly, thepresent invention relates to species specific probes originating fromthe Internal Transcribed Spacer (ITS) region of rDNA for the detectionof fungal species such as Candida albicans, Candida parapsilosis,Candida tropicalis, Candida kefyr, Candida krusei, Candida glabrata,Candida dubliniensis, Aspergillus flavus, Aspergillus versicolor,Aspergillus nidulans, Aspergillus fumigatus, Cryptococcus neoformans andPneumocystis carinii in clinical samples, and methods using said probes.

BACKGROUND OF THE INVENTION

Fungal infections are becoming an increasingly significant cause ofmorbidity and mortality. In the course of the 1980s, the rate ofbloodstream infection by Candida albicans surged by 48%. Patients atparticular risk of mycoses are those with diminished immune defenses—notonly organ transplant patients or those receiving intensive treatmentfor cancer, but also diabetics or people with indwelling catheters.

Fungal infections account for a large number of AIDS-defining diagnosesand complicate the course of most patients with HIV disease. The ECHOestimates that there will be more than 20 million HIV-infected adultsalive in the year 2000. The impact of fungi disease in AIDS patients isimmense because of their recurring nature. Of the 25 conditions whichmake up the case definition for AIDS, seven are caused by fungalpathogens. Of the most frequent AIDS indicator diseases occurring amonghomosexual/bisexual men, 70% were fungal infections, with Pneumocystiscarinii pneumonia (PCP) being the most common of all diseases.

Fungi occur in a wide variety of forms, from yeasts (single-celledorganisms which reproduce by budding) and moulds (which occur in longfilaments known as hyphae) to the dimorphic fungi which have achameleon-like ability to behave as yeasts in one environment and mouldin another.

Of the many different types of fungi only a few have the potential tocause disease, and the severity of their effects varies widely.Superficial mycoses, caused by a fungus such as Epidermophyton,Microsporum, Trichophyton or Sporothrix growing on the body surface(skin, nails or hair) are unpleasant but usually mild infections.

Deep mycoses, involving the internal organs, are often life-threatening.The fungi Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus,Histoplasma are responsible for deep mycoses, and pose tremendouschallenges for clinicians. Clinically, candidiasis and aspergillosisaccount for between 80% and 90% of systemic fungal infections inimmunocompromised patients. Recently, infections caused by Pneumocystiscarinii are more frequently found in AIDS patients.

Pneumocystis carinii is a major opportunistic infectious agent inimmunocompromised patients, causing pneumonia which has a high mortalityrate if the patient is not properly treated (Stringer J. R., 1996).Therefore, timely diagnosis of P. carinii pneumonia (PCP) is criticalfor patient management. Currently, diagnosis of PCP is usually made bymorphological demonstration of organisms in bronchoalveolar lavage (BAL)fluid, induced sputum, or specimens obtained by open lung biopsy (Creganet al., 1990). Although morphological diagnosis is rapid, it requireshighly experienced personnel and good specimens. Given the inability toculture P. carinii in vitro, molecular biology-based methods have beenused for the detection of this pathogen and to study P. cariniiepidemiology (Lu et al., 1995).

Candidoses comprise a range of human opportunistic infections which mayoccur in either acute or chronic forms. Candida infections frequentlyarise on the mucosal surfaces of the mouth or vagina. Chronichyperplastic candidosis of the oral mucosa is of particular importancesince it has been associated with the development of squamous cellcarcinoma. In addition to superficial lesions, deeper candidainfections, such as esophagitis and endocarditis, may occur,particularly in immunocompromised individuals (Heimdahl et al., 1990).

In the past, many studies of candidosis have not identified candidalisolates to species level. Indeed, in the case of chronic hyperplasticcandidosis, reporting is usually limited to the presence of structuresconsistent with candidal hyphae following histological examination oflesional tissue. However, it is becoming increasingly recognized thatboth species and subspecies of Candida differ in their ability to causedisease. Traditional methods used for the identification and typing ofclinical isolates of Candida include morphological and biochemicalanalysis (Williamson et al., 1986), colony morphotyping (Soll, D. R.1992), resistogram typing (Sobczak, H., 1985), and serotyping (Brawner,D. L., 1991). These techniques are time-consuming, and their reliance onphenotypic expression makes them potentially unreliable. An alternativemethod would be one based on genotypic properties. Genotypic methodshave been used for the detection and typing of Candida strains(Bart-Delabesse et al., 1993; Holmes et al., 1994), but have been usedless frequently for differentiation of species.

Other medically important Candida species, next to the most frequentlyisolated pathogen C. albicans, are C. glabrata. C. krusei and C.tropicalis. The latter species are much less susceptible to classicalantifungal drugs.

The genus Cryptococcus contains many species, wherein Cryptococcusneoformans is considered the only human pathogen. Initial cryptococcalinfection begins by inhalation of the fungus into the lungs, usuallyfollowed by hematogeneous spread to the brain and meninges. Involvementof the skin, bores, and joints is seen, and Cryptococcus neoformans isoften cultured from the urine of patients with disseminated infection.In patients without HIV infection, cryptococcosis, particularlycryptococcal meningitis, usually is seen in association with underlyingconditions such as lupus erythematosis, sarcoidosis, leukemia,lymphomas, and Cushing's syndrome (Chuck et al., 1989).

Cryptococcosis is one of the defining diseases associated with AIDS.Patients with cryptococcosis and serologic evidence of HIV infectionsare considered to have AIDS. In nearly 45% of AIDS patients,cryptococcosis was reported as the first AIDS-defining illness. Becausenone of the representing signs or symptoms of cryptococcal meningitis(such as headache, fever, and malaise) are sufficiently characteristicto distinguish it from other infections that occur in patients withAIDS, determining cryptococcal antigen titers and culturing blood andcerebrospinal fluid are useful in making a diagnosis (Chuck et al.1989).

The clinical diagnosis of pulmonary cryptococcosis in patients withoutunderlying diseases is generally difficult. Since the diagnosis is oftenestablished only by examination of tissue obtained from lung biopsyspecimens, other more sensitive and specific methods are needed for thesimple and fast detection of the fungus. One such approach involves thedetection of fungal antigens in serum (Kohno et al., 1993). However,evaluations of serological assays for detecting cryptococcal antigenshowed false-positive reactions with sera from patients infected withTrichosporon beigelii (Kohno et al., 1993).

Aspergillosis is now considered the second most common fungal infectionrequiring hospitalization. In patients with positive fungus cultures,Aspergillus species are the second most common isolate after Candidaspecies (Goodwin et al., 1992). The pathological responses caused bymembers of the genus Aspergillus vary in severity and clinical courseand may occur as both primary and secondary infections (Rinaldi, M. G.,1988).

Invasive aspergillosis (IA) is a life-threatening condition inimmunocompromised patients, particularly those treated with chemotherapyfor hematologic malignancies or those receiving high-dosecorticosteroids (Fisher et al., 1981). An early diagnosis ofaspergillosis is of great importance because early treatment may resolvethis potentially fatal infection. Unfortunately, the diagnosis of IAremains difficult and sometimes is confirmed only at autopsy. Atpresent, a firm diagnosis is established by histological examination oftissue samples obtained during open lung biopsy as well as by detectingthe causative pathogenic fungi in clinical samples. Serological testssuch as those involving the detection of antibodies for Aspergillusspecies are less helpful because of the poor antibody responses inimmunocompromised patients. In addition, the methods used for detectingcirculating Aspergillus antigens, such as radioimmunoassay,immunoblotting assay, enzyme immunoassay, and the latex agglutinationtest, have poor sensitivity (Rogers et al., 1990; Sabetta et al., 1985).

Recently, for the diagnosis of IA, PCR has been used to detect DNAspecific for Aspergillus species in bronchoalveolar lavage (BAL) fluidfrom patients with IA (Bretage et al., 1995).

Laboratory diagnosis of fungal infections is often problematic. Fungiare often difficult to culture from readily accessible samples, such aspatient's urine, blood or sputum. And because fungi are ubiquitous innature, a single positive culture from urine or sputum is of limitedclinical value. The possibility of contamination is always a very realconsideration when interpreting laboratory results. A finding ofAspergillus in sputum, for example, is in isolation of limited value andmust be evaluated in the context of the patient's clinical signs andsymptoms. Often tissue biopsies (from lung or brain) are needed fordefinitive diagnosis and blood cultures should be carried out for allpatients. Isolation of yeast—such as Candida—from blood is highlypredictive of invasive fungal disease. However Candida is cultured fromblood in less than 20% of patients with disseminated candidiasis.

Because of the limitation of culture techniques many researchers havetried to find specific antibodies against Candida and Aspergillus insequence by using the titre of antibodies as diagnostic criterium.However, the sensitivity of these assays is very low—often less than50%—as many immunocompromised patients have difficulty raising anadequate immune response. Because of its poor sensitivity,immunodiagnosis of fungal infection is not cost-effective. It oftengives a false negative result, and may also lead to fungal infectionsbeing diagnosed where none exist, leading to the inappropriateadministration of antifungal drugs.

Because of the shortcomings of antibody detection, much attention hasbeen directed to tests which detect fungal antigens or metabolites inbody fluids. A major problem is the transient nature of antigens inserum. For most antigen detection tests the overall sensitivity isunacceptably low, although multiple serum sampling somewhat improvesdetection of antigenaemia.

Moreover, none of the above-cited methods allows, the identification ofthe fungus up to the species level. Efficient treatment regimens offungal diseases require a correct identification of the fungus at thespecies level. For example, certain Candida species such as C. glabrataand C. krusei are less susceptible to the classical azole drugs.

The diagnostics of mycoses is an area where there is a great need fornew sophisticated techniques. As already seen in virology and to somedegree in bacteriology, the use of specific DNA probes, accompanied byDNA or RNA amplification systems, for the diagnosis of fungal infectionmay prove useful, and may revolutionize laboratory diagnosis andmanagement of patients with serious fungal disease.

Recently, several methods for detection of fungal pathogens using DNAtechnology have been described. Genes encoding the rRNA, especially the18S and 28S rRNA genes, have been frequently used as a target fordeveloping species specific probes (e.g. U.S. Pat. No. 5,827,651;Einsele et al. 1997). Others report on the use of the InternalTranscribed Spacer (ITS) region, located in between the 18S and 28S rRNAgenes, as a target region for the specific detection of fungi. Lu et al.(1995) describe the subtyping of Pneumocystis carinii strains usingprobes originating from the ITS region. Williams et al. (1995)demonstrate identification of Candida species by PCR amplification andrestriction length polymorphism analysis of the ITS amplified regions.Kumeda and Asao (1996) use PCR amplification and single strandconformation polymorphism (sscp) analysis of the ITS region todifferentiate species of Aspergillus. U.S. Pat. No. 5,693,501 describesspecific primers originating from the ITS-1 region for detection ofHistoplasma capsulatum. A number of patent applications (WO98/50584;WO95/29260; U.S. Pat. No. 5,814,463; U.S. Pat. No. 5,955,274) describedetection and differentiation of different plant fungal pathogens basedon specific amplification of or hybridization to ITS-region sequences.

Detection of Candida species based on ITS-2 region sequences has beendescribed by several groups. Fujita et al. (1995) describe ITS-2 probesfor different Candida species and methods for detection anddifferentiation after a general amplification step with universalprimers ITS3 and ITS4. Elie et al. (1998) and several related patentapplications (WO98/11257; WO99/06596; U.S. Pat. No. 5,426,027) describea set of 18 Candida species probes originating from the ITS-2 region.Shin et al. (1999) describe detection and differentation of threeCandida species in a single reaction tube, using amplification with theuniversal primers ITS3 and ITS4 and hybridization to ITS-2 probes.Botelho and Planta (1994) describe probes for Candida albicans, derivedfrom the ITS-1 or ITS-2 regions. The ITS-2 region probes show a betterspecies specificity.

Species specific probes originating from the ITS-region of othermedically important fungal species, such as species belonging toAspergillus, Cryptococcus, or Pneumocystis have not been described yet.Moreover, methods for simultaneous detection and differentiation of awide variety of fungal species with clinical importance have not beendescribed yet. Such methods would provide an answer to the need forrapid, highly sensitive and species specific detection of fungalpathogens in clinical samples, allowing a quick installment of efficienttreatment regimens, and close monitoring of a patient's progression.

SUMMARY AND AIMS OF THE INVENTION

The present invention describes nucleic acid molecules (oligonucleotideprobes) hybridizing specifically with the ITS region of different fungalspecies with clinical importance. More particularely, probes aredescribed hybridizing selectively with the ITS-1 and/or ITS-2 region ofseveral Candida species, Aspergillus species, Cryptococcus neoformansand Pneumocystis carinii. The most preferred probes of the currentinvention are located in the ITS-1 region, which separates the 18S and5.8S rRNA genes.

In addition, methods are described for quick sensitive and specificdetection and differentiation of these fungal pathogenic species, inparticular, methods are described for the simultaneous detection anddifferentiation of different fungal species in one single hybridizationassay. Such multi parameter detection methods may be particularly usefulin the detection of opportunistic infections in patients with impairedimmunity systems, such as organ transplant patients, patients receivingintensive anticancer treatments, diabetics or AIDS patients.

The methods and probes described are useful tools in clinical diagnosisof fungal infections. Moreover, they may be used to monitor the diseaseand to guide an appropriate antifungal therapy. The probes and methodsdescribed may also be used as laboratory research tools to study thedifferent fungal organisms, and their phylogenetic relationship.

The fungal species detected and identified by the probes and methods ofthe invention include Candida albicans, Candida parapsilosis, Candidatropicalis, Candida kefyr, Candida krusei, Candida glabrata, Candidadubliniensis, Aspergillus flavus, Aspergillus versicolor, Aspergillusnidulans, Aspergillus fumigatus, Cryptococcus neoformans andPneumocystis carinii. The methods of the current invention allow todetect any of the aforementioned species either alone, or in combinationwith each other, depending on the set of probes applied in the method.All probes are designed such that they are functional under identicalhybridization conditions, thus allowing any possible combination. Theparticular set of probes combined in a given method may depend onseveral ad hoc parameters, such as: the type of sample (respiratorytract, urogenitary tract, gastrointestinal tract, cerebrospinal fluid,blood samples, skin or tissue biopsies . . . ), the clinical symptoms ofthe patient, the desired level of specificity (genus, species, strain),the type of application (screening assay, confirmation assay, therapymonitoring, research tool for strain characterization, epidemiology . .. ).

It is thus an object of the current invention to provide nucleic acidprobes and primers for the specific detection and identification ofseveral fungal pathogens of clinical importance.

More particularly, it is an object of the current invention to providenucleic acid probes hybridizing specifically to the ITS (InternalTranscribed Spacer) region of different fungal pathogens. The probesdisclosed in the current invention hybridize to the ITS-1 or ITS-2region, most preferably to the ITS-1 region.

In particular, it is an object of the current invention to provideprobes for the detection and identification of several Candida species,including C. albicans, C. parapsilosis, C. tropicalis, C. kefyr, C.krusei, C. glabrata and C. dubliniensis, of several Aspergillus species,including A. flavus, A. versicolor, A. nidulans and A. fumigatus, ofCryptococcus neoformans and Pneumocystis carinii.

In addition, it is an object of the current invention to provide quick,sensitive and specific methods to detect and identify these fungalpathogens in clinical samples or in cultures.

It is also an object of the current invention to provide for a quick andefficient treatment method of blood samples, resulting in a release ofthe nucleic acids from fungal cells present in the blood sample, and aremoval of possible PCR inhibitors present in the blood sample.

It is furthermore an object of the present invention to provide methodswhich enable a simultaneous detection and identification of these fungalspecies possibly present in a sample, in one single assay.

It is also an aim of the present invention to provide methods forclinical diagnosis, monitoring and therapeutic managing of fungaldiseases.

These and other objects, including specific advantages and features ofthe current invention will become apparent in the detailed descriptionof the several embodiments hereafter.

DEFINITIONS

The term “probe” refers to isolated single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence to be detected. Complementarity of the probe sequence tothe target sequence is essential and complete for the central part ofthe probe (=core of the probe), where no mismatches to the targetsequence are allowed. Towards the extremities (3′ or 5′) of the probe,minor variations in the probe sequence may sometimes occur, withoutaffecting the species specific hybridization behaviour of the probe. The“core sequence” of the probe is the central part, and represents morethan 70%, preferably more than 80%, most often more than 90% of thetotal probe sequence.

The probes of the current invention specifically hybridize to the fungalspecies for which they are designed. This species specific hybridizationbehaviour will be illustrated amply; in the examples section. Throughoutthis invention, the sequences of the probes are always represented fromthe 5′ end to the 3′ end. They are represented as single stranded DNAmolecules. It should be understood however that these probes may also beused in their RNA form (wherein T is replaced by U), or in theircomplementary form.

The probes of the current invention may be formed by cloning ofrecombinant plasmids containing inserts comprising the correspondingnucleotide sequences, if need be by cleaving the latter out from thecloned plasmids upon using the adequate nucleases and recovering them,e.g. by fractionation according to molecular weight. The probesaccording to the present invention can also be synthesized chemically,for instance by the conventional phospho-triester method.

Preferably, the probes of the current invention have a length from about10 to about 30 nucleotides. Variations are possible in the length of theprobes and it should be clear that, since the central part of the probeis essential for its hybridization characteristics, possible deviationsof the probe sequence versus the target sequence may be allowabletowards head and tail of the probe, especially when longer probesequences are used. These variant probes, which may be conceived fromthe common knowledge in the art, should however always be evaluatedexperimentally, in order to check if they result in equivalenthybridization characteristics than the original probes.

The term “isolated” as used herein means that the oligonucleotides ofthe current invention are isolated from the environment in which theynaturally occur. In particular, it means that they are not an % morepart of the genome of the respective fungal species, and thus liberatedfrom the remaining flanking nucleotides in the ITS region of said fungalspecies. On the contrary, new (=heterologous) flanking regions may beadded to the 3′ and/or 5′ end of the probe, in order to enhance itsfunctionality. Functional characteristics possibly provided by saidheterologous flanking sequences are e.g. ease of attachment to a solidsupport, ease of synthesis, ease of purification, labelling functionetc.

The term “complementary” nucleic acid as used in the current inventionmeans that the nucleic acid sequences can form a perfect base-paireddouble helix with each other.

The term “species specific hybridization” refers to a selectivehybridization of the probes of the invention to the nucleic acids of thespecies to be detected (=target organism), and not to nucleic acidsoriginating from strains belonging to other species (=non-targetorganisms). Species specific hybridization in the context of the presentinvention also implies a selective hybridization of the probes of theinvention to the ITS region (=target region) of the organism to bedetected, and limits occasional “random” hybridization to other genomicsequences. Species specificity is a feature which has to beexperimentally determined. Although it may sometimes be theoreticallypredictable, species specificity can only refer to those non-targetorganisms which have been tested experimentally.

The term “primer” refers to an isolated single stranded oligonucleotidesequence capable of acting as a point of initiation for synthesis of aprimer extension product which is complementary to the nucleic acidstrand to be copied. The length and the sequence of the primer must besuch that they allow to prime the synthesis of the extension products.Preferably the primer is about 5-50 nucleotides long, more preferablyfrom 10 to 40 nucleotides long. Specific length and sequence will dependon the complexity of the required DNA or RNA targets, as well as on theconditions of primer use such as temperature and ionic strength.

The oligonucleotides used as primers or probes may also comprisenucleotide analogues such as phosphorothiates (Matsukura et al., 1987),alkylphosphorothiates (Miller et al. 1979) or peptide nucleic acids(Nielsen et al., 1991; Nielsen et al., 1993) or may containintercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the originalDNA sequences of the invention, these variations will necessitateadaptions with respect to the conditions under which the oligonucleotideshould be used to obtain the required specificity and sensitivity.However the eventual results of hybridisation will be essentially thesame as those obtained with the unmodified oligonucleotides.

The introduction of these modifications may be advantageous in order topositively influence characteristics such as hybridization kinetics,reversibility of the hybrid-formation, biological stability of theoligonucleotide molecules, etc.

The term “sample” represents any material possibly containing fungalnucleic acids, which may have to be released from the cells. Preferably,the term “sample” refers to a clinical sample, such as a sample takenfrom blood, from the respiratory tract (sputum, bronchoalveolar lavage(BAL)), from cerebrospinal fluid (CSF), from the urogenital tract(vaginal secretions, urine), from the gastrointestinal tract (saliva,faeces) or biopsies taken from organs, tissue, skin e.a. The term“sample” may also refer to a sample of cultured fungal cells, eithercultured in liquid medium or on solid growth media. Fungal DNA presentin said samples may be prepared or extracted according to any of thetechniques known in the art.

The term “ITS” is the abbreviated term for Internal Transcribed Spacerregion located between the 18S and the 28S rRNA genes in the rRNA operonof the fungal species' nuclear DNA (for a review, see White et al.1990). The ITS region is subdivided in the ITS-1 region, which separatesthe 18S and 5.8S rRNA genes, and the ITS-2 region which is found betweenthe 5.8S and 28S rRNA genes.

The “target” material in these samples may be either genomic DNA orprecursor ribosomal RNA of the organism to be detected (=targetorganism), or amplified versions thereof. These molecules are calledtarget nucleic acids. More specifically, the target nucleic acid in thegenome is the Internal Transcribed Spacer region (ITS). According to apreferred embodiment, the target region in the genome is the ITS-1region. The target sequence is that part of the ITS sequence which isfully complementary to the core part of the probe.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides in its most general form for a method todetect and identify fungal pathogenic species in a sample, comprising atleast the following steps:

(i) releasing, isolating and/or concentrating the nucleic acids of thefungal pathogens possibly present in the sample,

(ii) if necessary, amplifying the Internal Transcribed Spacer region(ITS) of said nucleic acids with at least one fungal universal primerpair,

(iii) hybridizing the nucleic acids of step (i) or (ii) with at leastone probe selected from the following group of species specificoligonucleotide probes:

GTCTAAACTTACAACCAATT, (SEQ ID NO 1) TGTCACACCAGATTATTACT, (SEQ ID NO 2)TATCAACTTGTCACACCAGA, (SEQ ID NO 3) GTAGGCCTTCTATATGGG, (SEQ ID NO 4)TGCCAGAGATTAAACTCAAC, (SEQ ID NO 5) GGTTATAACTAAACCAAACT, (SEQ ID NO 6)TTTTCCCTATGAACTACTTC, (SEQ ID NO 7) AGAGCTCGTCTCTCCAGT, (SEQ ID NO 8)GGAATATAGCATATAGTCGA, (SEQ ID NO 9) GAGCTCGGAGAGAGACATC, (SEQ ID NO 10)TAGTGGTATAAGGCGGAGAT, (SEQ ID NO 11) CTAAGGCGGTCTCTGGC, (SEQ ID NO 12)GTTTTGTTCTGGACAAACTT, (SEQ ID NO 13) CTTCTAAATGTAATGAATGT, (SEQ ID NO14) CATCTACACCTGTGAACTGT, (SEQ ID NO 15) GGACAGTAGAGAATATTGG, (SEQ ID NO16) GGACTTGGATTTGGGTGT, (SEQ ID NO 17) GTTTACTGTACCTTAGTTGCT, (SEQ ID NO18) CCGCCATTCATGGCC, (SEQ ID NO 19) CGGGGGCTCTCAGCC, (SEQ ID NO 20)CCTCTCGGGGGCGAGCC, (SEQ ID NO 21) CCGAGTGCGGCTGCCTC, (SEQ ID NO 22)CCGAGTGCGGGCTGC, (SEQ ID NO 23) GAGCCTGAATACCAAATCAG, (SEQ ID NO 24)GAGCCTGAATACAAATCAG, (SEQ ID NO 25) GTTGATTATCGTAATCAGT, (SEQ ID NO 26)GCGACACCCAACTTTATT, (SEQ ID NO 27) ATGCTAGTCTGAAATTCAAAAG, (SEQ ID NO28) GGATTGGGCTTTGCAAATATT, (SEQ ID NO 29) TTCGCTGGGAAAGAAGG, (SEQ ID NO30) GCTTGCCTCGCCAAAGGTG, (SEQ ID NO 31) TAAATTGAATTTCAGTTTTAGAATT, (SEQID NO 32) TTGTCACACCAGATTATTACTT, (SEQ ID NO 33)GGTTTATCAACTTGTCACACCAGA, (SEQ ID NO 34) GGTATCAACTTGTCACACCAGATT, (SEQID NO 35) GGTTATAACTAAACCAAACTTTTT, (SEQ ID NO 36)GGGAATATAGCATATAGTCGA, (SEQ ID NO 37) GGTTTTGTTCTGGACAAACTT, (SEQ ID NO38) CATCTACACCTGTGAACTGTTT, (SEQ ID NO 39) CCGACACCCAACTTTATTTTT, (SEQID NO 40) GTTGATTATCGTAATCAGTT, (SEQ ID NO 41) GAACTCTGTCTGATCTAGT, (SEQID NO 42) GTCTGAATATAAAATCAGTCA, (SEQ ID NO 43)

-   -   or variants of said probes, said variants differing from the        sequences cited above by the deletion and/or addition of one or        two nucleotides at the 5′ and/or 3′ extremity of the nucleotide        sequence, without affecting the species specific hybridization        behaviour of the probe,    -   or the RNA equivalents of said probes, wherein T is replaced by        U,    -   or the complementary nucleic acids of said probes, and

(iv) detecting the hybridization complexes formed in step (iii).

The probes used in the above described method for detection of fungalpathogens are all hybridizing to the Internal Transcribed Spacer regionof fungal species. More particularly, as illustrated further in theexamples section (see Table 1), the above-cited probes hybridizeselectively to the following target regions: ITS-1 region of Candidaalbicans (probes represented by SEQ ID NO 1, 2, 3, 33, 34, 35), ITS-1region of C. parapsilosis (probes represented by SEQ ID NO 4,5), ITS-1region of C. tropicalis (probes represented by SEQ ID NO 6, 36). ITS-1region of C. kefyr (probes represented by SEQ ID NO 7, 8), ITS-1 regionof C. krusei (probes represented by SEQ ID NO 9, 37), ITS-1 region of C.glabrata (probes represented by SEQ ID NO 10), ITS-1 region of C.dubliniensis (probes represented by SEQ ID NO 13, 38), ITS-2 region ofC. dubliniensis (probes represented by SEQ ID NO 11, 12), ITS-1 regionof Cryptococcus neoformans (probes represented by SEQ ID NO 14, 15, 16,39), ITS-2 region of Cryptococcus neoformans (probes represented by SEQID NO 17), ITS-1 region of Aspergillus flavus (probes represented by SEQID NO 18, 19, 20, 42), ITS-1 region Aspergillus versicolor (probesrepresented by SEQ ID NO 21, 43), ITS-1 region of Aspergillus nidulans(probes represented by SEQ ID NO 22, 23, 24, 25), ITS-1 region ofAspergillus fumigatus (probes represented by SEQ ID NO 26, 41), ITS-2region of Aspergillus fumigatus (probes represented by SEQ ID NO 27,40), ITS-1 region of Pneumocystis carinii (probes represented by SEQ IDNO 31, 32), and ITS-2 region of Pneumocystis carinii (probes representedby SEQ ID NO 28, 29, 30).

The expression “variants” of the probes encompasses probes representedby a variant sequence which differs from any of the sequences citedabove by the deletion and/or addition of 1 or 2 nucleotides at the 3′and/or 5′ extremity of the probe sequence, in so far as such deletion oraddition does not change the species specific character of therespective probe. It will be understood that the addition of 1 or 2nucleotides at the extremities of the probes will usually be done inaccordance with the sequence flanking the target sequence in the ITSregion from which the probe is isolated. This means that one shallnormally not choose “any” nucleotide to extend the probe sequence, butonly those nucleotides which are flanking the probe sequence in the ITSregion. The information about the flanking sequences of the probes caneasily be obtained by aligning the probe sequence to the ITS sequence.The ITS sequence itself may be obtained by sequencing the ITS region,after cloning or e.g. PCR amplification of the ITS region with fungaluniversal primer pairs, or may be retrieved from publicly availablesources.

The probes and variant probes as above described may also be extended atthe 3′ and/or 5′ end with non-ITS (=heterologous) flanking sequences.Without affecting the intrinsic hybridization behaviour of the probe,this heterologous tailing process may provide some additionalcharacteristics to the probe molecule, such as e.g. adhesion to asurface by polyT tailing, as described furtheron in the examplessection.

The above-described method may be applied for the detection of onesingle fungal species, so called “single analyte detection”, e.g. inmicrotiter plates, or for the detection of several fungal speciessimultaneously, so called “multi parameter detection”, e.g. in a LineProbe Assay (LiPA). The probes described have been selected such thatthey may all be functional (i.e. show the desired species specificity)under the same hybridization and wash conditions. This allows the methodto be used for the simultaneous detection and differentiation of severalfungal species in one single hybridization assay.

The term “fungal universal primer pair” means that the primer pairamplifies the Internal Transcribed Spacer region of most, if not all,fungal species. The sequences of “fungal universal primer pairs” arephylogenetically conserved in order to enable amplification withindifferent species of fungi. They are located in the rRNA genes flankingthe ITS region, i.e. in the 18S, 5.8S or 28S rRNA genes. Amplificationof the full ITS region, the ITS-1 or the ITS-2 region may be envisaged.“Fungal universal primer pairs” suitable for the methods described inthe current invention have been described by a. o. White et al. (1990).

In a preferred embodiment, the method described above includes anamplification step using a fungal universal primer pair which is chosenfrom the following group of primer pairs, as described by White et al.(1990):

ITS5 (forward): GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO:44) and ITS4(reverse): TCCTCCGCTTATTGATATGC, (SEQ ID NO:45) ITS5 (forward):GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO:44) and ITS2 (reverse):GCTGCGTTCTTCATCGATGC, (SEQ ID NO:46) ITS1 (forward): TCCGTAGGTGAACCTGCGG(SEQ ID NO:47) and ITS4 (reverse): TCCTCCGCTTATTGATATGC, (SEQ ID NO:45)ITS1 (forward): TCCGTAGGTGAACCTGCGG (SEQ ID NO:47) and ITS2 (reverse):GCTGCGTTCTTCATCGATGC, (SEQ ID NO:48) ITS3 (forward):GCATCGATGAAGAACGCAGC (SEQ ID NO:49) and ITS4 (reverse):TCCTCCGCTTATTGATATGC. (SEQ ID NO:45)

The full ITS region may be amplified using a combination of the ITS1 andITS4 primer, or the ITS-5 and ITS4 primer. The ITS-1 region may beamplified using a combination of the ITS1 and ITS2 primer, or the ITS5and ITS1 primer, while the ITS-2 region may be amplified using acombination of the ITS3 and ITS4 primer.

According to a preferred embodiment, amplification of the ITS-1 regionis envisaged, and probes will be chosen from the ITS-1 region. As willbe shown further on in the examples section, the current invention showsthat methods based on amplification of and hybridization to the ITS-1region usually show a higher sensitivity than methods based onamplification of and hybridization to the full ITS region, or the ITS-2amplified region. The present invention furthermore shows thatidentification and differentiation of most, if not all, of the differentfungal species listed can be accomplished by using probe sequencesoriginating from the ITS-1 region only.

Thus, according to a preferred embodiment, the present inventionprovides for a method for the detection and identification of fungalpathogenic species in a sample, comprising at least the following steps:

(i) releasing, isolating and or concentrating the nucleic acids of thefungal pathogens possibly present in the sample.

(ii) amplifying the ITS-1 region of said nucleic acids with at least oneof the following primer pairs according to s-Mite et al. (1990): (ITS5and ITS2) or (ITS1 and ITS2),

(iii) hybridizing the nucleic acids of step (i) or (ii) with at leastone probe selected from the following group of species specificoligonucleotide probes:

GTCTAAACTTACAACCAATT, (SEQ ID NO 1) TGTCACACCAGATTATTACT, (SEQ ID NO 2)TATCAACTTGTCACACCAGA, (SEQ ID NO 3) GTAGGCCTTCTATATGGG, (SEQ ID NO 4)TGCCAGAGATTAAACTCAAC, (SEQ ID NO 5) GGTTATAACTAAACCAAACT, (SEQ ID NO 6)TTTTCCCTATGAACTACTTC, (SEQ ID NO 7) AGAGCTCGTCTCTCCAGT, (SEQ ID NO 8)GGAATATAGCATATAGTCGA, (SEQ ID NO 9) GAGCTCGGAGAGAGACATC, (SEQ ID NO 10)GTTTTGTTCTGGACAAACTT, (SEQ ID NO 13) CTTCTAAATGTAATGAATGT, (SEQ ID NO14) CATCTACACCTGTGAACTGT, (SEQ ID NO 15) GGACAGTAGAGAATATTGG, (SEQ ID NO16) GTTTACTGTACCTTAGTTGCT, (SEQ ID NO 18) CCGCCATTCATGGCC, (SEQ ID NO19) CGGGGGCTCTCAGCC, (SEQ ID NO 20) CCTCTCGGGGGCGAGCC, (SEQ ID NO 21)CCGAGTGCGGCTGCCTC, (SEQ ID NO 22) CCGAGTGCGGGCTGC, (SEQ ID NO 23)GAGCCTGAATACCAAATCAG, (SEQ ID NO 24) GAGCCTGAATACAAATCAG, (SEQ ID NO 25)GTTGATTATCGTAATCAGT, (SEQ ID NO 26) GCTTGCCTCGCCAAAGGTG, (SEQ ID NO 31)TAAATTGAATTTCAGTTTTAGAATT, (SEQ ID NO 32) TTGTCACACCAGATTATTACTT, (SEQID NO 33) GGTTTATCAACTTGTCACACCAGA, (SEQ ID NO 34)GGTATCAACTTGTCACACCAGATT, (SEQ ID NO 35) GGTTATAACTAAACCAAACTTTTT, (SEQID NO 36) GGGAATATAGCATATAGTCGA, (SEQ ID NO 37) GGTTTTGTTCTGGACAAACTT,(SEQ ID NO 38) CATCTACACCTGTGAACTGTTT, (SEQ ID NO 39)GTTGATTATCGTAATCAGTT, (SEQ ID NO 41) GAACTCTGTCTGATCTAGT, (SEQ ID NO 42)GTCTGAATATAAAATCAGTCA, (SEQ ID NO 43)

-   -   or variants of said probes, said variants differing from the        sequences cited above by the deletion and/or addition of one or        two nucleotides at the 5′ and/or 3′ extremity of the nucleotide        sequence, without affecting the species specific hybridization        behaviour of the probe,    -   or the RNA equivalents of said probes, wherein T is replaced by        U,    -   or the complementary nucleic acids of said probes, and

(iv) detecting the hybridization complexes formed in step (iii).

Amplification of the nucleic acids may be carried out according to anymethod known in the art, including the polymerase chain reaction (PCR;Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988),nucleic acid sequence-based amplification (NASBA; Guatelli et al.,1990), transcription-based amplification system (TAS; Kwoh et al.,1989), strand displacement amplification (SDA; Duck, 1990) oramplification by means of Qβ replicase (Lomeli et al., 1989) or anyother suitable method to amplify nucleic acid molecules know-n in theart.

Amplification of the nucleic acids to be detected has of course theadvantage of increasing the sensitivity of detection. Moreover, usingfungal universal primer pairs, methods for simultaneous detection can bedeveloped including simultaneous amplification of the ITS region ofseveral fungal species, followed by species specific hybridization.Amplification also allows the incorporation of a label into theamplified nucleic acids, which opens different ways of detecting thehybridization complexes formed, and which may increase the sensitivityof detection again. Labelling may be carried out by the use of labellednucleotides incorporated during the polymerase step of the amplificationsuch as illustrated by e.g. Bej et al. (1990) or labelled primers, or byany other method known to the person skilled in the art. The nature ofthe label may be isotopic (³²P, ³⁵S, etc.) or non-isotopic (biotin,digoxigenin, fluorescein etc.). Alternatively, of course; the probes ofthe invention may be labelled.

Hybridization of the nucleic acids is carried out according to standardmethods. Preferably, stringent hybridization conditions are used. i.e.conditions enabling differentiation by hybridization between nucleicacids which differ by only one single nucleotide. Examples of stringentconditions applicable to the probes of the current invention are ahybridization buffer of 2×SSC (Sodium Saline Citrate) and 0.1% SDS at ahybridization temperature of 50° C. All probes of the current inventionare designed such that they show the desired (=species specific)hybridization behaviour at stringency conditions defined by ahybridization medium of 2×SSC and 0.1% SDS and a hybridizationtemperature of 50° C. Any other hybridization condition (i.e. any othercombination of hybridization buffer and hybridization temperature)resulting in the same degree of stringency is of course also suitablefor the probes of the current invention. The design of hybridizationconditions to meet certain stringency criteria is common knowledge inthe art of hybridization.

Hybridization may be carried out in solution or on a solid support, witheither the probes being immobilized to the solid support or the nucleicacids to be detected being immobilized. Immobilization of the nucleicacids to a solid support may be done covalently, or using non-covalentbinding forces.

According to a preferred embodiment, the oligonucleotide probes of theinvention are immobilized to a solid support, and reverse hybridizationis carried out.

The term “solid support” in the current invention refers to anysubstrate to which an oligonucleotide probe can be coupled, providedthat its hybridization characteristics are retained and provided thatthe background of hybridization remains low. Usually the solid supportwill be a microtiter plate, a membrane (e.g. nylon or nitrocellulose) ora microsphere (bead). Prior to application to the membrane or fixationit may be convenient to modify the nucleic acid probe in order tofacilitate fixation or to improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH2 groups, SH groups,carboxylic groups, or coupling with biotin, haptens or proteins.

According to a preferred embodiment, the oligonucleotides used in theabove described methods of detection are immobilized to a solid supportby means of a homopolymer tailing sequence (e.g. polyT) which is addedat the 3′ or 5′ extremity of the probe. Said tailing may be done duringchemical synthesis of the oligonucleotide, usually resulting in a 5′polyT tail, or afterwards, e.g. via enzymatic tailing using terminaldeoxynucleotidyl transferase (Pharmacia), resulting in a 3′ polyT tail.

Detection of the hybridization complexes formed may be done according toany methods known in the art, the type of detection of course dependingon the type of label used. When biotin is used as a label, streptavidinconjugated detection agents may be used, such as e.g. streptavidinconjugated alkaline phosphatase, causing a blue precipitation signal, orstreptavidin conjugated horse raddish peroxidase, causing a colorreaction in solution.

According to a special embodiment, the invention provides for a LiPA(Line Probe Assay) method for detecting and identifying fungal pathogensin a sample, as shown below in the examples section. LiPA is a reversehybridization assay using oligonucleotide probes immobilized as parallellines on a solid support strip (as described by Stuyver et al. (1993)and WO 94/12670). LiPA is particularly advantageous since it is fast andsimple to perform. Moreover, this method is amenable to automation(Auto-LiPA, Innogenetics, Zwijnaarde, Belgium) and thus particularlysuitable for clinical settings where multiple samples can be processedsimultaneously. It is to be understood however, that any other type ofhybridization assay or format using any of the selected probes asdescribed further, is also covered by the present invention.

According to a preferred embodiment, the method described above may beapplied for the simultaneaous detection and differentiation of fungalpathogens present in a particular type of sample. For example, when thesample is a blood or serum sample, the methods of the invention maycomprise detection and differentiation of Candida species, Aspergillusspecies, and Cryptococcus species. When the sample originates from therespiratory tract (e.g. sputum samples, BAL samples) the methods of thecurrent invention may comprise detection and differentiation of Candidaspecies, most often species other than Candida albicans. Aspergillusspecies, Cryptococcus species and Pneumocystis carinii. When the sampleoriginates from CSF, the methods of the invention may comprise detectionand differentiation of Candida species, Aspergillus species,Cryptococcus species and Pneumocystis carinii. When the sampleoriginates from skin or wound tissue, the methods of the invention maycomprise detection and differentiation of Aspergillus species, Candidaspecies, Cryptococcus species. When the sample originates from theurogenital tract, the methods of the invention enable detection anddifferentiation of different types of Candida species.

According to a more specific embodiment, the current invention providesfor a method as described above, wherein said fungal pathogen is aCandida species, and wherein the probes of step (iii) are chosen fromamong SEQ ID NO 1, 2, 3, 33, 34 and 35 for C. albicans, SEQ ID NO 4 and5 for C. parapsilosis, SEQ ID NO 6 and 36 for C. tropicalis. SEQ ID NO 7and 8 for C. kefyr, SEQ ID NO 9 and 37 for C. krusei, SEQ ID NO 10 forC. glabrata, and SEQ ID NO 11, 12, 13 and 38 for C. dubliniensis.

According to a more specific embodiment, the current invention providesfor a method to detect Candida albicans in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 1, 2, 3, 33, 34 and 35, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C.albicans.

According to another specific embodiment, the current invention providesfor a method to detect Candida parapsilosis in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 4 and 5, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C.parapsilosis.

According to another specific embodiment, the current invention providesfor a method to detect Candida tropicalis in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 6 and 36, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C.tropicalis.

According to another specific embodiment, the current invention providesfor a method to detect Candida kefyr in a sample, said method including

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 7 and 8, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C. kefyr.

According to another specific embodiment, the current invention providesfor a method to detect Candida krusei in a sample, said method including

(i) hybridizing, the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 9 and 37, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C. krusei.

According to another specific embodiment, the current invention providesfor a method to detect Candida glabrata in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to a proberepresented by SEQ ID NO 10, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C.glabrata.

According to another specific embodiment, the current invention providesfor a method to detect Candida dubliniensis in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 11, 12, 13 and 38 and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C.dubliniensis.

According to a further embodiment, the current invention provides for amethod to detect and identify fungal pathogenic species as describedabove, wherein said fungal pathogen is an Aspergillus species, andwherein the probes of step (iii) are chosen from among SEQ ID NO 18, 19,20 and 42 for A. flavus, SEQ ID NO 21 and 43 for A. versicolor, SEQ IDNO 22, 23, 24 and 25 for A. nidulans, and SEQ ID NO 26, 27, 40 and 41for A. fumigatus.

According to a more specific embodiment, the current invention providesfor a method to detect Aspergillus flavus in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 18, 19, 20 and 42, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of A. flavus.

According to another specific embodiment, the current invention providesfor a method to detect Aspergillus versicolor in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 21 and 43, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of A.versicolor.

According to another specific embodiment, the current invention providesfor a method to detect Aspergillus nidulans in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 22, 23, 24 and 25, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of A.nidulans.

According to another specific embodiment, the current invention providesfor a method to detect Aspergillus fumigatus in a sample, said methodincluding

(i) hybridizing the nucleic acids present in the sample to at least oneof the probes represented by SEQ ID NO 26, 27, 40 and 41, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of A.fumigatus.

According to another particular embodiment, the present inventionprovides for a method to detect Cryptococcus neoformans in a sample,including

(i) hybridization of the nucleic acids present in the sample to at leastone of the probes represented by SEQ ID NO 14, 15, 16, 17, and 39 and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of C.neoformans.

According to another particular embodiment, the present inventionprovides for a method to detect Pneumocystis carinii in a sample,including

(i) hybridization of the nucleic acids present in the sample to at leastone of the probes represented by SEQ ID NO 28, 29, 30, 31 and 32, and

(ii) detecting the hybridization complexes formed, the presence of saidhybridization complexes being indicative for the presence of P. carinii.

According to a preferred embodiment, the oligonucleotide probes used inthe above described methods of detection are immobilized to a solidsupport.

According to a particularly preferred embodiment, the current inventionprovides for a method for detection and identification of fungalpathogens in a sample as described above, whereby the amplification ofstep (ii) is mandatory, and includes the labelling of the nucleic acidsto be detected.

According to a particularly advantageous embodiment, the currentinvention provides for a method for the simultaneous detection anddifferentiation of at least two fungal pathogenic species in one singleassay, including

(i) releasing, isolating and/or concentrating the nucleic acids of thefungal pathogens possibly present in the sample,

(ii) amplifying the Internal Transcribed Spacer region (ITS) of saidnucleic acids with at least one fungal universal primer pair,

(iii) hybridizing the nucleic acids of step (i) or (ii) with at leasttwo of the following species specific oligonucleotide probes:

GTCTAAACTTACAACCAATT (SEQ ID NO 1) TGTCACACCAGATTATTACT (SEQ ID NO 2)TATCAACTTGTCACACCAGA (SEQ ID NO 3) GTAGGCCTTCTATATGGG (SEQ ID NO 4)TGCCAGAGATTAAACTCAAC (SEQ ID NO 5) GGTTATAACTAAACCAAACT (SEQ ID NO 6)TTTTCCCTATGAACTACTTC (SEQ ID NO 7) AGAGCTCGTCTCTCCAGT (SEQ ID NO 8)GGAATATAGCATATAGTCGA (SEQ ID NO 9) GAGCTCGGAGAGAGACATC (SEQ ID NO 10)TAGTGGTATAAGGCGGAGAT (SEQ ID NO 11) CTAAGGCGGTCTCTGGC (SEQ ID NO 12)GTTTTGTTCTGGACAAACTT (SEQ ID NO 13) CTTCTAAATGTAATGAATGT (SEQ ID NO 14)CATCTACACCTGTGAACTGT (SEQ ID NO 15) GGACAGTAGAGAATATTGG (SEQ ID NO 16)GGACTTGGATTTGGGTGT (SEQ ID NO 17) GTTTACTGTACCTTAGTTGCT (SEQ ID NO 18)CCGCCATTCATGGCC (SEQ ID NO 19) CGGGGGCTCTCAGCC (SEQ ID NO 20)CCTCTCGGGGGCGAGCC (SEQ ID NO 21) CCGAGTGCGGCTGCCTC (SEQ ID NO 22)CCGAGTGCGGGCTGC (SEQ ID NO 23) GAGCCTGAATACCAAATCAG (SEQ ID NO 24)GAGCCTGAATACAAATCAG (SEQ ID NO 25) GTTGATTATCGTAATCAGT (SEQ ID NO 26)GCGACACCCAACTTTATT (SEQ ID NO 27) ATGCTAGTCTGAAATTCAAAAG (SEQ ID NO 28)GGATTGGGCTTTGCAAATATT (SEQ ID NO 29) TTCGCTGGGAAAGAAGG (SEQ ID NO 30)GCTTGCCTCGCCAAAGGTG (SEQ ID NO 31) TAAATTGAATTTCAGTTTTAGAATT (SEQ ID NO32) TTGTCACACCAGATTATTACTT, (SEQ ID NO 33) GGTTTATCAACTTGTCACACCAGA,(SEQ ID NO 34) GGTATCAACTTGTCACACCAGATT, (SEQ ID NO 35)GGTTATAACTAAACCAAACTTTTT, (SEQ ID NO 36) GGGAATATAGCATATAGTCGA, (SEQ IDNO 37) GGTTTTGTTCTGGACAAACTT, (SEQ ID NO 38) CATCTACACCTGTGAACTGTTT,(SEQ ID NO 39) CCGACACCCAACTTTATTTTT, (SEQ ID NO 40)GTTGATTATCGTAATCAGTT, (SEQ ID NO 41) GAACTCTGTCTGATCTAGT, (SEQ ID NO 42)GTCTGAATATAAAATCAGTCA, (SEQ ID NO 43)

-   -   or variants of said probes, said variants differing from the        sequences cited above by the deletion and/or addition of one or        two nucleotides at the 5′ and/or 3′ extremity of the nucleotide        sequence, without affecting the species specific hybridization        behaviour of the probe,    -   or the RNA equivalents of said probes, wherein T is replaced by        U,    -   or the complementary nucleic acids of said probes,    -   wherein said probes have been immobilized to a solid support on        specific locations,

(iv) detecting the hybridization complexes formed in step (iii),

(v) identifying the species present in the sample by the location of thehybridization signal on the solid support.

In the above-cited embodiment, the probes of the invention areimmobilized on a solid support on specific locations, e.g. as discreteparallel lines or spots on a membrane strip, or in different wells of amicrotiter plate. Each location contains a determined amount of at leastone species specific probe, and can therefore be considered as a“species specific location”. If necessary, several probes hybridizing tothe same species may be combined on one single location. By locating thehybridization signal obtained on the solid support, it is thus possibleto identify the fungal species present in the sample (step (v) of theabove described method).

According to a preferred embodiment, as will be shown in the examplessection, the immobilization of the probes to the solid support occursvia the non-covalent binding of a polyT tail which is attached to one ofthe extremities of the probe. If enzymatic tailing occurs, the polyTtail is added at the 3′ end. If synthetic tailing (SGS) occurs, thepolyT tail is usually added at the 5′ end. Both types of tailing may beapplied to the probes of the invention. If a different type of tailingresults in a different hybridization behaviour of the probe, theoligonucleotide sequence may have to be adapted slightly. Examples ofsuch slight variations are illustrated in Table 1 furtheron, where acertain type of probe may be mentioned twice: e.g. Calb2 (SEQ ID NO 2)and Calb2 (SGS) (SEQ ID NO 33), the former probe mentioned beingfunctional with a 3′ polyT tail, the latter probe being functional witha 5′ polyT tail.

According to another embodiment, the current invention also provides foran isolated oligonucleotide molecule having a nucleotide sequencerepresented by any of the sequences SEQ ID NO 1 to 43, or variants ofsaid probes, said variants differing from the sequences cited above bythe deletion and/or addition of one or two nucleotides at the 5′ and/or3′ extremity of the nucleotide sequence, without affecting the speciesspecific hybridization behaviour of the probe, or the RNA equivalents ofsaid probes, wherein T is replaced by U, or the complementary nucleicacids of said probes.

Preferred nucleic acids of the invention consist of a nucleotidesequence represented by any of the sequences SEQ ID NO 1-43. Asmentioned above, addition and or deletion of 1 or 2 nucleotides at the5′ and/or 3′ extremity may result in functional equivalent molecules,which are also encompassed by the current invention. Moreover, as setout above, it may be desirable to modify the nucleic acid probe in orderto facilitate fixation or to improve the hybridization efficiency (e.g.homopolymer tailing, coupling with different reactive groups . . . ).Such modified nucleic acid molecules are also part of the currentinvention.

More in particular, the current invention provides for an isolatedoligonucleotide molecule as described above for use as a speciesspecific primer or probe in the detection of one of the following fungalpathogenic species: Candida albicans, Candida parapsilosis, Candidatropicalis, Candida kefyr, Candida krusei, Candida glabrata, Candidadubliniensis, Aspergillus flavus, Aspergillus versicolor, Aspergillusnidulans, Aspergillus fumigatus, Cryptococcus neoformans or Pneumocystiscarinii.

The current invention also provides for a method as described above,wherein the sample is a blood sample, and wherein step (i) includes

-   -   incubation of the blood sample with lysis buffer (10 mM        Tris-HCl, ph 7.5, 10 mM EDTA, 50 mM NaCl), followed by        centrifugation and removal of the supernatant, and    -   vortexing of the resuspended cell pellet in the presence of        glass beads.

The above-described sample pretreatment is described in further detailin the examples section, and is shown to be applicable to a wide varietyof fungal species.

FIGURE LEGEND

FIG. 1: Hybridization results of full ITS, ITS-1 or ITS-2 amplicons toLiPA strips containing species-specific probes.

Lanes:

lane 1: PCR blanc (i.e. no PCR amplicon added to this lane)

lanes 2,3: C. albicans (2: full ITS, 3: ITS-1)

lanes 4,5: C. parapsilosis (4: full ITS, 5: ITS-1)

lanes 6,7: C. glabrata (6: full ITS, 7: ITS-1)

lanes 8,9: C. tropicalis (8: full ITS, 9: ITS-1)

lanes 10,11: C. krusei (10: full ITS, 11: ITS-1)

lanes 12,13: C. dubliniensis (12: full ITS, 13: ITS-2)

lanes 14,15: Cr. neoformans (14: full ITS, 15: ITS-1)

lanes 16,17: A. fumigatus (16: full ITS, 17: ITS-1)

lanes 18: A. nidulans (full ITS)

lane 19: A. flavus (full ITS)

lane 20: oligo-dA-bio (hybridizes to (poly-T) tail of the immobilizedprobes)

Probe Lines:

colour reaction control line (heterologous biotinylated oligonucleotide)

row 1: Calb1

row 2: Calb2

row 3: Calb3

row 4: Cpara2

row 5: Cglab

row 6: Ctrop

row 7: Ckrus

row 8: Cdub1

row 9: Cdub2

row 10: Cmeo2

row 11: Cmeo4

row 12: Afum1

row 13: Afum2

row 14: Anid1

row 15: Afla1

row 16: Aver

row 17: Aver

FIG. 2: LiPA evaluation on clinical isolates

lanes 1-15: different clinical isolates, identified as

lanes 1-4, 6, 7, 10: C. albicans

lane 5: C. glabrata

lane 8, 9, 11, 12, 14, 15: A. fumigatus

lane 13: A. flavus

rows: see FIG. 1

TABLE 1 ITS probe sequences for fungal detection and differentiationProbe Sequence SEQ ID NO Organism Spacer region Calb1GTCTAAACTTACAACCAATT SEQ ID NO 1 Candida albicans ITS1 Calb2TGTCACACCAGATTATTACT SEQ ID NO 2 Candida albicans ITS1 Calb2 (SGS)TTGTCACACCAGATTATTACTT SEQ ID NO 33 Candida albicans ITS1 Calb3TATCAACTTGTCACACCAGA SEQ ID NO 3 Candida albicans ITS1 Calb3 (SGS1)GGTTTATCAACTTGTCACACCAGA SEQ ID NO 34 Candida albicans ITS1 Calb3 (SGS2)GGTATCAACTTGTCACACCAGATT SEQ ID NO 35 Candida albicans ITS1 Cpara1GTAGGCCTTCTATATGGG SEQ ID NO 4 Candida parapsilosis ITS1 Cpara2TGCCAGAGATTAAACTCAAC SEQ ID NO 5 Candida parapsilosis ITS1 CtropGGTTATAACTAAACCAAACT SEQ ID NO 6 Candida tropicalis ITS1 Ctrop (SGS)GGTTATAACTAAACCAAACTTTTT SEQ ID NO 36 Candida tropicalis ITS1 Ckef1TTTTCCCTATGAACTACTTC SEQ ID NO 7 Candida kefyr ITS1 Ckef2AGAGCTCGTCTCTCCAGT SEQ ID NO 8 Candida kefyr ITS1 CkrusGGAATATAGCATATAGTCGA SEQ ID NO 9 Candida krusei ITS1 Ckrus (SGS)GGGAATATAGCATATAGTCGA SEQ ID NO 37 Candida krusei ITS1 CglabGAGCTCGGAGAGAGACATC SEQ ID NO 10 Candida glabrata ITS1 Cdub11TAGTGGTATAAGGCGGAGAT SEQ ID NO 11 Candida dubliniensis ITS2 Cdub12CTAAGGCGGTCTCTGGC SEQ ID NO 12 Candida dubliniensis ITS2 Cdub13GTTTTGTTCTGGACAAACTT SEQ ID NO 13 Candida dubliniensis ITS1 Cdub13 (SGS)GGTTTTGTTCTGGACAAACTT SEQ ID NO 38 Candida dubliniensis ITS1 Crneo1CTTCTAAATGTAATGAATGT SEQ ID NO 14 Cryptococcus neoformans ITS1 Crneo2CATCTACACCTGTGAACTGT SEQ ID NO 15 Cryptococcus neoformans ITS1 Crneo2(SGS) CATCTACACCTGTGAACTGTTT SEQ ID NO 39 Cryptococcus neoformans ITS1Crneo3 GGACAGTAGAGAATATTGG SEQ ID NO 16 Cryptococcus neoformans ITS1Crneo4 GGACTTGGATTTGGGTGT SEQ ID NO 17 Cryptococcus neoformans ITS2Afla1 GTTTACTGTACCTTAGTTGCT SEQ ID NO 18 Aspergillus flavus ITS1 Afla2CCGCCATTCATGGCC SEQ ID NO 19 Aspergillus flavus ITS1 Afla3CGGGGGCTCTCAGCC SEQ ID NO 20 Aspergillus flavus ITS1 Afla4GAACTCTGTCTGATCTAGT SEQ ID NO 42 Aspergillus flavus ITS1 AverCCTCTCGGGGGCGAGCC SEQ ID NO 21 Aspergillus versicolor ITS1 Aver3 (SGS)GTCTGAATATAAAATCAGTCA SEQ ID NO 43 Aspergillus versicolor ITS1 Anid1CCGAGTGCGGCTGCCTC SEQ ID NO 22 Aspergillus nidulans ITS1 Anid1ACCGAGTGCGGGCTGC SEQ ID NO 23 Aspergillus nidulans ITS1 Anid2GAGCCTGAATACCAAATCAG SEQ ID NO 24 Aspergillus nidulans ITS1 Anid2AGAGCCTGAATACAAATCAG SEQ ID NO 25 Aspergillus nidulans ITS1 Afum2GTTGATTATCGTAATCAGT SEQ ID NO 26 Aspergillus fumigatus ITS1 Afum2 (SGS)GTTGATTATCGTAATCAGTT SEQ ID NO 41 Aspergillus fumigatus ITS1 Afum1GCGACACCCAACTTTATT SEQ ID NO 27 Aspergillus fumigatus ITS2 Afum1 (SGS)CCGACACCCAACTTTATTTTT SEQ ID NO 40 Aspergillus fumigatus ITS2 Pear1ATGCTAGTCTGAAATTCAAAAG SEQ ID NO 28 Pneumocystis carinii ITS2 Pear2GGATTGGGCTTTGCAAATATT SEQ ID NO 29 Pneumocystis carinii ITS2 Pear3TTCGCTGGGAAAGAAGG SEQ ID NO 30 Pneumocystis carinii ITS2 Pear4GCTTGCCTCGCCAAAGGTG SEQ ID NO 31 Pneumocystis carinii ITS1 Pear5TAAATTGAATTTCAGTTTTAGAATT SEQ ID NO 32 Pneumocystis carinii ITS1

TABLE 2 Hybridization results obtained with a selection of the probes ofthe invention applied on a wide variety of fungal species. Species Ref.nr Calb1 Calb2 Calb3 Cpara2 Cglab Ctrop Ckrus Cdubl1 Penicillium speciesP. aurantiogriseum PIL 563 P. antiogriseum PIL 333 var. melanoconidiumP. expansum PIL 346 P. verucosum PIL 781 P. verucosum PIL 115 P.verucosum PIL 25 P. hordei PIL 351 P. islanidcum PIL 778 P. marlensiiPIL 9 P. ruber PIL 162 Aspergillus species A. fumigatus NCPF 7097 A.fumigatus NCPF 2109 A. fumigatus NCPF 2937 A. nidulans NCPF 7063 A.nidulans PIL 272 A. niger NCPF 2828 A. niger NCPF 2599 A. niger PIL 4 A.restriclus PIL 167 A. restriclus PIL 87 A. restriclus PIL 34 A.restriclus PIL 116 A. ochraceus PIL 253 A. candidus PIL 129 A. terreusPIL 422 A. flavus NCPF 2199 A. flavus PIL 444 A. flavus PIL 512 A.flavus PIL 447 A. flavus PIL 345 A. flavus PIL 378 A. flavus PIL 295 A.flavus PIL 499 A. flavus PIL 480 A. flavus PIL 377 A. flavus PIL 110 A.versicolor PIL 347 A. versicolor PIL 725 A. versicolor PIL 564 A.versicolor PIL 565 A. versicolor PIL 293 A. versicolor PIL 399 A.versicolor PIL 656 A. versicolor PIL 576 A. versicolor PIL 770 Eurotiumspecies E. amstelodami PIL 218 E. chevalieri PIL 280 Fusarium species F.avenicum PIL 569 F. culmorum PIL 772 F. culmorum PIL 234 F. graminearumPIL 210 F. poae PIL 773 F. moniliforme PIL 450 Candida species C.albicans NCPF 3302 + + + C. albicans NCPF 3328 + + + C. albicans NCPF3345 + + + C. albicans NCPF 3822 + + + C. albicans NCPF + + + C.guillermondii 44490 C. krusei NCPF 3896 + C. krusei NCPF 3922 + C.krusei NCPF 3845 + C. parapsilosis NCPF 3847 + C. glabrata NCPF 3872 +C. glabrata NCPF 3700 + C. kefyr NCPF 3863 C. lusitaniae NCPF 3898 C.tropicalis NCPF 3924 + C. dubliniensis NCPF 3870 + CD 36 Cryptococcusspecies C. neoformans NCPF 3756 var. gatti C. neoformans NCPF 3232 var.neoformans C. laurentii NCPF 3836 C. albidus NCF 3147 Other speciesAltemaria alternata PIL 764 Monascus tuber PIL 48 Species Cdubl2 Crneo2Crneo4 Afum1 Afum2 Anid Afla Aver Penicillium species P. aurantiogriseumP. antiogriseum var. melanoconidium P. expansum P. verucosum P.verucosum P. verucosum P. hordei P. islanidcum P. marlensii P. ruberAspergillus species A. fumigatus + + A. fumigatus + + A. fumigatus + +A. nidulans + A. nidulans + A. niger A. niger A. niger A. restriclus A.restriclus A. restriclus A. restriclus A. ochraceus A. candidus A.terreus A. flavus + A. flavus + A. flavus + A. flavus + A. flavus + A.flavus + A. flavus + A. flavus + A. flavus + A. flavus + A. flavus + A.versicolor + A. versicolor + A. versicolor + A. versicolor + A.versicolor + A. versicolor + A. versicolor + A. versicolor + A.versicolor + Eurotium species E. amstelodami E. chevalieri Fusariumspecies F. avenicum F. culmorum F. culmorum F. graminearum F. poae F.moniliforme Candida species C. albicans C. albicans C. albicans C.albicans C. albicans C. guillermondii C. krusei C. krusei C. krusei C.parapsilosis C. glabrata C. glabrata C. kefyr C. lusitaniae C.tropicalis C. dubliniensis + Cryptococcus species C. neoformans + + var.gatti C. neoformans + + var. neoformans C. laurentii C. albidus Otherspecies Altemaria alternata Monascus tuber (+) = positive hybridizationsignal, ( ) = no signal detected

EXAMPLES 1. Specificity Testing of the Oligonucleotide Probes

1.1. Nucleic Acid Extraction.

A rapid extraction method based on physical disruption of the fungalcells followed by crude separation of the cell debris from the genomicDNA was used for the production of DNA from single colonies of yeasts(Roberts, 1997). For filamentous fungi, a more elaborate samplepreparation method based on a combination of beadbeating and lysis witha GuSCN buffer followed by capturing of the DNA on silica was used.

1.2. PCR Amplification

20-50 ng genomic DNA or 5 μl of DNA extracted by the rapid extractionprocedure described above were included in the PCR reaction. PCRreactions contained per 100 μl reaction: 200 μM of each dNTP's, 1×Taqbuffer, 3 mM MgCl2, 15% glycerol, 40 pmol of each biotinylated primer(ITS5 and ITS4 for amplification of the full ITS region, ITS5 and ITS2for amplification of ITS-1 only), 1 U Uracil N glycosylase and 2.5 U Taqpolymerase. PCR thermal cycling conditions were the following: 95° C.for 10 min for 1 cycle (hotstart), 94° C. for 30 sec., 55° C. for 30sec., 72° C. for 2 min. for 30 cycles, and a final extension at 72° C.for 10 min. for 1 cycle.

1.3. Production of LiPA Strips

Synthetic probes were provided enzymatically at the 3′ end with apoly-T-tail using terminal deoxynucleotidyl transferase (Pharmacia) in astandard reaction buffer. After one hour incubation, the reaction wasstopped and the tailed probes were precipitated and washed with ice-coldethanol. The oligonucleotide probe sequences of the invention areindicated in Table 1.

If tailing occurred by chemical synthesis, the poly-T-tail was attachedat the 5′ end of the oligonucleotide probe. Chemically tailed probes mayneed a small modification (deletion and/or addition of a few nucleotidesat one or both of the extremities) of the probe sequence as compared tothe enzymatically tailed probe, in order to show comparablehybridization characteristics. The chemically tailed modified probes areindicated with the extension “(SGS)” in Table 1 and, if the modificationis an addition of nucleotides, it is indicated in bold.

Probes were dissolved in 6×SSC at their respective specificconcentrations and applied as horizontal lines on membrane strips.Biotinylated DNA was applied alongside as positive control. Theoligonucleotides were fixed to the membrane by baking at 80° C. for 12hours. The membrane was than sliced into 4 mm strips.

1.4. LiPA Test Performance

Equal volumes (5 to 10 μl) of the biotinylated PCR fragments and of thedenaturation solution (400 mM NaOH/10 mM EDTA) were mixed in testtroughs and incubated at room temperature for 5 min. Subsequently, 2 mlof the 50° C. prewarmed hybridization solution (2×SSC/0.1% SDS) wasadded followed by the addition of one strip per test trough.Hybridization occurred for 1 hour at 50° C. in a closed shaking waterbath. The strips were washed twice with 2 ml of stringent was solution(2×SSC, 0.1% SDS) at room temperature for 20 sec., and once at 50° C.for 15 min. Following this stringent wash, strips were rinsed two timeswith 2 ml of the Innogenetics standard Rinse Solution (RS). Strips wereincubated on a rotating platform with the alkaline phosphatase-labelledstreptavidin conjugate, diluted in standard Conjugate Solution (CS) for3 min. at room temperature. Strips were then washed twice with 2 ml ofRS and once with standard Substrate Buffer (SB), and the colour reactionwas started by adding BCIP and NBT to the SB. After 30 min. at roomtemperature, the colour reaction was stopped by replacing the colourcompounds by distilled water. Immediately after drying, the strips wereinterpreted. The complete procedure described above can also be replacedby the standard Inno-LiPA automation device (Auto-LiPA. InnogeneticsN.V., Zwijnaarde, Belgium). The above mentioned buffers (RS, CS, SB) mayall be obtained from Innogenetics N.V. (Zwijnaarde, Belgium).

1.5. Hybridization Results

Specific hybridization results for a set of probes of Table 1 aresummarized in Table 2. Species-specificity occurs when all the strainsbelonging to the respective fungal species show positive hybridization(−) and none of the other fungal species tested shows cross reactionwith the species-specific probes under the hybridization conditionsused.

FIG. 1 represents an example of LiPA strips. By comparing lanes 3, 9,11, 15 and 17 with respectively lanes 2, 8, 10, 14 and 16 it is clearthat the sensitivity of detection is higher (i.e. stronger hybridizationsignal obtained) when only the ITS-1 region is amplified, as compared toamplification of the full ITS region.

2. Sensitivity Testing of the PCR Amplification

2.1. Nucleic Acid Extraction

Small modifications were made on large-scale DNA extraction methods asdescribed in literature (Holmes et al. 1994, Nho et al. 1997, Weig etal. 1997) and used to extract genomic DNA from Candida, Cryptococcus andAspergillus species yielding DNA of high purity suitable for long termstorage. DNA concentrations of the prepared stocks were calculated basedon optical density measurements.

2.2. Sensitivity Testing

Amplification experiments were performed on dilution series of genomicDNA (ranging from 50 or 100 ng to 50 or 100 fg) using differentprimersets, either combination of ITS5/ITS2 amplifying the ITS-1 regiononly or ITS5/ITS4 amplifying the fall ITS region. Amplicons generatedwere visualised on ethidium-bromide stained gels and hybridized to LiPAstrips containing the appropriate probes.

2.3. Results

Table 3 below summarizes the detection limits obtained with dilutionseries of genomic DNA isolated from the following organisms: C.albicans, C. neoformans and A. fumigatus.

Organism Amplification Agarosegel LiPA Hybridization C. albicans ITS1 10pg 100 fg Full ITS 1 pg 100 fg C. neoformans ITS1 100 pg 1 pg Full ITS10 pg 10 pg A. fumigatus ITS1 500 fg 50 fg Full ITS 5 pg 5 pg

Amplification of the full ITS region results in a better sensitivitywhen amplicons are visualized on ethidiumbromide stained agarose gels(except for A. fumigatus). Inadequate staining of small amplicons couldexplain this phenomenon.

When hybridized to probes immobilized on LiPA strips (see e.g. FIG. 1),it is clearly seen that amplification of the ITS-1 region (smalleramplicon) results in a more sensitive detection limit compared to fullITS amplification. LiPA was able to specifically detect C. albicans DNAdown to 100 fg, Cryptococcus neoformans DNA down to 1 pg, and A.fumigatus DNA down to 50 fg.

3. Sensitivity Testing in Spiked Sputum Samples

3.1. Nucleic Acid Extraction

200 μl sputum specimens were spiked with dilutions of C. albicans cells(10⁵-1 cells/ml) or decreasing amounts (not quantifiable) of A.fumigatus mycelia. Fungal DNA was isolated from these sputum specimensusing a modification of the method described by Boom et al., 1990. Theoverall time taken to process 15 sputum specimens was 2 hours.

3.2. Amplification

5 μl of the extracted DNA was used as target material in the PCR (100μl). PCR reactions were carried out as described above. ITS-1amplification was performed using primer combination ITS5+ITS2 andamplification of the full ITS region was performed using ITS5 and ITS4primers.

3.3. Results

Following LiPA hybridization of the obtained amplicons a detection limitof 10-100 cells/ml sputum was obtained for C. albicans. Amplification ofspiked sputum specimens with A. fumigatus mycelia resulted in adetection of the lowest level of spiked A. fumigatus mycelium.

4. Sensitivity Testing in Spiked Blood Samples

Evaluation of different methods for the extraction of fungal DNA fromblood has previously been reported in the literature (Loffler et al.1997). Most of the methods evaluated involved known enzymatic approacheswith some modifications. However, these methods failed to consistentlyyield high quality DNA resulting in variation in the detection limitsachieved between experiments (data not shown). It was also noted thatDNA extracted from A. fumigatus using these methods required a “hotstart” PCR approach for sensitive PCR amplification of the extracted DNA(data not shown). As a result, a new sample pretreatment method forblood samples was developed by the current invention, as describedbelow.

4.1. Nucleic Acid Extraction

200 μl, 1 ml and 5 ml blood samples were inoculated with decreasingconcentrations of C. albicans cells (10⁵-10¹ cells). The inoculatedblood samples were pre-treated to lyse and remove the red blood cells.Blood samples of 200 μl were lysed in 800 μl of lysis buffer (10 mMTris-HCl, [pH 7.5], 10 mM EDTA, 50 mM NaCl) at room temperature for 10minutes and centrifuged at 13,000 rpm for 5 min, the supernatantdiscarded and the pellet was resuspended in 100 μl of sterile H₂0. Bloodsamples of 1 ml and 5 were lysed in 3 mls of lysis buffer andcentrifuged at 2000 rpm for 10 mins, the supernatant discarded and thepellet was resuspended in 100 μl of sterile H₂0. Glass beads (0.5 mmzirconium glass beads stored in 0.2% SDS) were added to the resuspendedpellet, the sample was vortexed in a mini-bead beater at top speed for190 seconds in order to mechanically disrupt the yeast cells. The DNAwas subsequently extracted using known methods such as e.g. described byBoom et al. (1999) or the QIAmp Tissue kit (Qiagen, Los Angeles,Calif.).

This same method proved to be applicable also to other fungal species,such as Aspergillus and Cryptococcus spp.

4.2. Amplification

PCR amplification of the ITS region was performed in a final volume of100 μl with 20 μl of DNA extracted from the blood samples (for DNAextracted from 5 ml blood samples, 20 μl of a 1/10 dilution is includedin the POR reaction) added to the PCR reaction containing a finalconcentration 0.25 mM deoxynucleotidetriphosphates (DU/dNTP's [2:1]), 1×reaction buffer (Promega, USA), 3 mM MgCl₂, 1 unit Uracil DNAglycosylase (Longo et al 1990; Roche-Boehringer Mannheim, Germany), 40pmol each of the forward ITS5 primer (5′-GAAAGTAAAAGTCGTAACAAGG-3′) (SEQID NO:50) and reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (SEQ IDNO:45), 2.5 units of Taq polymerase (Promega, USA), made to a finalvolume of 100 μl in nuclease free water (Sigma-Aldrich Ltd, UK).

PCR amplification was performed in a Touchdown™ Thermocycler (Hybaid,UK), with the following cycling conditions: 37° C. for 10 minutes for 1cycle followed by 94° C. for 2 minutes for 1 cycle followed by 40 cyclesof DNA denaturation at 94° C. for 30 seconds, primer annealing at 55° C.for 30 seconds and DNA extension at 72° C. for 2 minutes, with a finalextension cycle at 72° C. for 10 minutes. 50 ng of C. albicans DNAextracted as described above was included as a positive control in thePCR reaction along with a no template negative control in each PCR run.

4.3. LiPA Hybridization Results and Evaluation of the Test Performance.

Following LiPA hybridization of the obtained amplicons, a detectionlimit of 2-10 cells/ml blood was consistently obtained for Candidaalbicans (results not shown). The PCR-LiPA and associated extraction offungal DNA from blood samples can be performed in a single working daywhile the technology itself may be easily integrated into a clinicaltesting laboratory that is already engaged in PCR as it does not requireany specialised equipment.

Early reports of PCR-based assays for the detection of Candida in blooddescribe hemi-nested approaches (Rand et al. 1994) or rely on cumbersomelaboratory-based techniques for post-PCR detection (Holmes et al., 1994;Jordan et al. 1994) which are unsuitable for large scale screening ofsamples. More recent reports describe a number of different approachesfor the detection of fungal pathogens in blood. These include a broadspectrum PCR-based approach (Van Burik et al., 1998) designed to detectthe presence of fungal infection in blood without identification of thespecific infectious agent but with a detection sensitivity of 4 cells/mlfollowing hybridisation with a non-radioactive pan-fungal DNA probe. Theauthors describe the successful application of this assay to thedetection of fungal infection in samples from a group of bone marrowtransplant patients. A microtitre-plate based assay for the detection ofthe five most clinically significant Candida species has also beendescribed (Shin-Ichi et al. 1995) and while they report a detectionsensitivity of 2 cells/200 μl of blood, the assay technology may be alittle cumbersome as the post-PCR hybridisation of the amplicons to thespecies-specific probes is performed in microcentrifuge tubes and thentransferred to a microtitre-plate for the detection step. A more recentpublication (Hee Shin et al. 1999) describes a very elegant assay forthe detection of up to three Candida species in a single reaction tubeby using DNA probes labelled with different fluorescent tags. This assayrepresents a two-step system, with the PCR amplification and post-PCRdetection being performed in a single tube and reducing the assay timefrom 7 hours to 5 hours. The authors describe the application of theassay technology for the detection of Candida in blood culture bottlespositive for the presence of fungal infection.

The PCR-LiPA assay described in the current invention is also amulti-parameter test as a single LiPA membrane includes DNA probes forthe detection of a wide range of different Candida species. It thereforehas the potential to detect and identify mixed Candida infections.Moreover, a universal approach for the preparation of fungal DNA fromCandida. Cryptococcus and Aspergillus ssp. in blood and/or respiratoryspecimens has been developed, as described above (see 4.1).

5. Testing of Clinical Isolates

Fungal DNA extraction was performed on cultures of clinical isolatesusing the methods as described above. PCR reactions were carried out asdescribed above and primer combination ITS5+ITS4 was used for full ITSamplification.

LiPA strips for fungal pathogens were evaluated on 100 clinicalisolates. The full ITS region was amplified from 75 clinical isolates ofdimorphic yeasts following the application of the rapid preparationmethod (Roberts et al. 1997) from a single colony. The full ITS regionwas amplified from 25 clinical isolates of filamentous fungi, followingpreparation of the DNA using the modified Boom et al. method.

Hybridization of the obtained PCR products to the LiPA strips identified2 isolates as C. parapsilosis, 2 isolates as C. glabrata, 15 isolates asA. fumigatus, 3 isolates as A. nidulans, 3 isolates as A. flavus and 5unidentified isolates (=belonging to other fungal species for which noprobes were present on the LiPA strip, but for which an amplicon wasobtained during the fungal-universal PCR reaction). All remainingisolates were identified as C. albicans and these were confirmed using abiochemical diagnostic kit “Murex C. albicans” (Murex Diagnostica). Anexample of the LiPA hybridization results obtained with 15 clinicalisolates is shown on FIG. 2.

The above results convincingly show that the probes of the currentinvention are not only applicable for the detection of laboratory fungalstrains, from which they were originally designed, but that they detectwith high specificity and sensitivity clinically occurring strains ofthose fungal species. The methods described in the current inventionwill therefore greatly facilitate the detection of fungal pathogens inclinical samples, if desirable in one single assay.

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1. An isolated oligonucleotide molecule consisting of SEQ ID NO: 26, 41,or the RNA equivalents of said SEQ IDs wherein T is replaced by U, orthe complements thereof.
 2. A method to detect and identify at least oneAspergillus species in one single assay, including (i) releasing,isolating and I or concentrating the nucleic acids of the fungalpathogens possibly present in the sample, (ii) optionally, amplifyingthe Internal Transcribed Spacer region (ITS) of said nucleic acids withat least one fungal universal primer pair, (iii) hybridising the nucleicacids of step (i) or (ii) with at least one oligonucleotide molecule ofclaim 1, (iv) detecting the hybridisation complexes formed in step(iii), and (v) identifying the Aspergillus species present in saidsample, based on the hybridisation complex formed.
 3. Method accordingto claim 2, wherein said fungal universal primer pair is chosen from thefollowing group of primer pairs: ITS5: GGAAGTAAAAGTCGTAACAAGG (SEQ IDNO: 44) and ITS4: TCCTCCGCTTATTGATATGC, (SEQ ID NO: 45) ITS5:GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO: 44) and ITS2: GCTGCGTTCTTCATCGATGC,(SEQ ID NO: 46) ITS1: TCCGTAGGTGAACCTGCGG (SEQ ID NO: 47) and ITS4:TCCTCCGCTTATTGATATGC, (SEQ ID NO: 45) ITS1: TCCGTAGGTGAACCTGCGG (SEQ IDNO: 47) and ITS2: GCTGCGTTCTTCATCGATGC, (SEQ ID NO: 46) ITS3:GCATCGATGAAGAACGCAGC (SEQ ID NO: 49) and ITS4: TCCTCCGCTTATTGATATGC.(SEQ ID NO: 45)


4. Method according to claim 2 wherein the Aspergillus species is A.fumigatus and wherein the at least one oligonucleotide molecule of step(iii) is chosen from among SEQ ID NOs:26 and
 41. 5. Method according toclaim 2 wherein the at least one oligonucleotide molecule of step (iii)is immobilized to a solid support.
 6. Method according to claim 2,further enabling the detection and identification of at least one of thefollowing fungal pathogens: Candida albicans, Candida parapsilosis,Candida tropicalis, Candida kefyr, Candida krusei, Candida glabrata,and/or Candida dubliniensis, wherein the nucleic acids of step (i) or(ii) are further hybridized with at least one of the following speciesspecific oligonucleotide probes: SEQ ID NOs:2 to 13, and 33 to
 38. 7.Method for the simultaneous detection and differentiation of at leasttwo Aspergillus species in one single assay, including (i) releasing,isolating and I or concentrating the nucleic acids of the fungalpathogens possibly present in the sample, (ii) optionally, amplifyingthe Internal Transcribed Spacer region (ITS) of said nucleic acids withat least one fungal universal primer pair, (iii) hybridising the nucleicacids of step (i) or (ii) with at least two oligonucleotide moleculeswherein at least one of the oligonucleotide molecules is anoligonucleotide of claim 1 and at least one of the oligonucleotides isselected from the group consisting of SEQ ID NOs: 18-27 and 40-43, underthe same hybridization conditions, (iv) detecting the hybridisationcomplexes formed in step (iii), and identifying the Aspergillus speciespresent in said sample, based on the hybridisation complex formed. 8.Method according to claim 7, wherein said fungal universal primer pairis chosen from the following group of primer pairs: ITS5:GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO: 44) and ITS4: TCCTCCGCTTATTGATATGC,(SEQ ID NO: 45) ITS5: GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO: 44) and ITS2:GCTGCGTTCTTCATCGATGC, (SEQ ID NO: 46) ITS1: TCCGTAGGTGAACCTGCGG (SEQ IDNO: 47) and ITS4: TCCTCCGCTTATTGATATGC, (SEQ ID NO: 45) ITS1:TCCGTAGGTGAACCTGCGG (SEQ ID NO: 47) and ITS2: GCTGCGTTCTTCATCGATGC, (SEQID NO: 48) ITS3: GCATCGATGAAGAACGCAGC (SEQ ID NO: 49) and ITS4:TCCTCCGCTTATTGATATGC. (SEQ ID NO: 45)


9. A combination of at least two isolated oligonucleotide moleculesconsisting of at least one oligonucleotide molecule of claim 1 and anucleotide sequence represented by any of SEQ ID NOs: 2 to 25, 27 to 40,42 and 43, or the RNA equivalents of said SEQ IDs wherein T is replacedby U, or the complementary nucleic acid of said SEQ IDs, wherein said atleast two oligonucleotide molecules are functional as hybridizationprobes under identical hybridization conditions.